CIRCLE-Seq for Interrogation of Off-Target Gene Editing.

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing.

Highlights of Gene and Cell Therapy for Epidermolysis Bullosa and Ichthyosis.

Advancements in the molecular genetics of epidermolysis bullosa (EB) and ichthyosis, two rare inherited skin conditions, have enabled the identification of genetic variants that cause these diseases. Alongside technological advancements in genetic medicine, the identification of variants causal of these rare skin conditions has led to preclinical research and the clinical development of various in vivo and ex vivo gene and cell therapies for their treatment. Gene and cell therapies are considered to be the most advanced forms of personalized medicine, demonstrating safety and efficacy in numerous rare diseases.

The Development of an Advanced Model for Multilayer Human Skin Reconstruction In Vivo.

Human skin reconstruction on immune-deficient mice has become indispensable for in vivo studies performed in basic research and translational laboratories. Further advancements in making sustainable, prolonged skin equivalents to study new therapeutic interventions rely on reproducible models utilizing patient-derived cells and natural three-dimensional culture conditions mimicking the structure of living skin. Here, we present a novel step-by-step protocol for grafting human skin cells onto immunocompromised mice that requires low starting cell numbers, which is essential when primary patient cells are limited for modeling skin conditions.

A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa.

Background: Gene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies.

Methods: We developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7).

Cell differentiation modifies the p53 transcriptional program through a combination of gene silencing and constitutive transactivation.

The p53 transcription factor is a master regulator of cellular responses to stress that is commonly inactivated in diverse cancer types. Despite decades of research, the mechanisms by which p53 impedes tumorigenesis across vastly different cellular contexts requires further investigation. The bulk of research has been completed using in vitro studies of cancer cell lines or in vivo studies in mouse models, but much less is known about p53 action in diverse non-transformed human tissues.

Extracellular vesicles released by human retinal pigment epithelium mediate increased polarised secretion of drusen proteins in response to AMD stressors.

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown.

Common molecular mechanisms of SLC6A1 variant-mediated neurodevelopmental disorders in astrocytes and neurons.

Solute carrier family 6 member 1 (SLC6A1) is abundantly expressed in the developing brain even before the CNS is formed. Its encoded GABA transporter 1 (GAT-1) is responsible for the reuptake of GABA into presynaptic neurons and glia, thereby modulating neurotransmission. GAT-1 is expressed globally in the brain, in both astrocytes and neurons.

Differentiating Induced Pluripotent Stem Cells Toward Mesenchymal Stem/Stromal Cells.

Differentiating human induced pluripotent stem cells (iPSCs) into multipotent mesenchymal stem/stromal cells (MSCs) offers a renewable source of therapeutically invaluable cells. However, the process of MSC derivation from iPSCs suffers from an undesirably low efficiency. In this chapter, we present an optimized procedure to produce MSCs from human iPSCs with a high efficiency.

A High-Efficiency Method for the Production of Endothelial Cells from Human Induced Pluripotent Stem Cells.

Endothelial cells (ECs) are important components of the circulatory system. These cells can be used for in vitro modeling of cardiovascular diseases and in regenerative medicine to promote vascularization of engineered tissue constructs. However, low proliferative capacity and patient-to-patient variability limit the use of primary ECs in the clinic and disease modeling.

Peptide-Functionalized Hydrogels Modulate Integrin Expression and Stemness in Adult Human Epidermal Keratinocytes.

The extracellular matrix (ECM) controls keratinocyte proliferation, migration, and differentiation through β-integrin signaling. Wound-healing research requires expanding cells in vitro while maintaining replicative capacity; however, early terminal differentiation under traditional culture conditions limits expansion. Here, a design of experiments approach identifies poly(ethylene glycol)-based hydrogel formulations with mechanical properties (elastic modulus, E = 20.

Generation of an Induced Pluripotent Stem Cell Line with the Constitutive EGFP Reporter.

The discovery of induced pluripotent stem cell (iPSC) technology has provided a versatile platform for basic science research and regenerative medicine. With the rise of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems and the ease at which they can be utilized for gene editing, creating genetically modified iPSCs has never been more advantageous for studying both organism development and potential clinical applications. However, to better understand the behavior and true therapeutic potential of iPSCs and iPSC-derived cells, a tool for labeling and monitoring these cells in vitro and in vivo is needed.

Efficient RNA-Based Reprogramming of Disease-Associated Primary Human Fibroblasts into Induced Pluripotent Stem Cells.

Reprogramming a patient's somatic cells into induced pluripotent stem cells (iPSCs) holds great promise for disease modeling and the development of autologous cellular therapeutics. However, it remains challenging to consistently reprogram primary human cells, as they are frequently aged, diseased, or in low abundance. Here we present a modified highly efficient and clinically relevant RNA-based method for reprogramming disease-associated and other difficult-to-reprogram human primary fibroblast lines into iPSCs.

Dynamic mechanical loading and growth factors influence chondrogenesis of induced pluripotent mesenchymal progenitor cells in a cartilage-mimetic hydrogel.

Human induced pluripotent stem cells (iPSCs) have emerged as a promising alternative to bone-marrow derived mesenchymal stem/stromal cells for cartilage tissue engineering. However, the effect of biochemical and mechanical cues on iPSC chondrogenesis remains understudied. This study evaluated chondrogenesis of induced pluripotent mesenchymal progenitor cells (iPS-MPs) encapsulated in a cartilage-mimetic hydrogel under different culture conditions: free swelling versus dynamic compressive loading and different growth factors (TGFβ3 and/or BMP2).

Gene Therapy for Skin Fragility Diseases: The New Generation.

Ex vivo gene therapy is a promising approach to treat devastating skin fragility diseases. March et al. and Takashima et al.

Generation of a Full-Thickness Human Skin Equivalent on an Immunodeficient Mouse.

Human skin equivalents composed of epidermal cells and fibroblasts are important for modeling human epidermal development, testing new therapeutics, and designing novel treatment strategies for human skin diseases. Here, we describe a procedure for the generation of an in vivo full-thickness human skin equivalent on an immunodeficient mouse using a grafting chamber system. The protocol involves mixing human epidermal cells and fibroblasts in a silicone grafting chamber that is surgically inserted onto the muscle fascia of a recipient immunodeficient mouse.

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach.

The N-peptide-binding mode is critical to Munc18-1 function in synaptic exocytosis.

Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to -ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A key SNARE-binding mode of SM proteins involves the N-terminal peptide (N-peptide) motif of syntaxin, a SNARE subunit localized to the target membrane. In membrane fusion assays, inhibition of N-peptide motif binding previously has been shown to abrogate the stimulatory function of Munc18-1, a SM protein involved in synaptic exocytosis in neurons.

High-efficiency RNA-based reprogramming of human primary fibroblasts.

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNA-367/302s delivered as mature miRNA mimics greatly enhances the reprogramming of human primary fibroblasts into iPSCs. This synergistic activity is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to human primary fibroblasts.

Folate dietary insufficiency and folic acid supplementation similarly impair metabolism and compromise hematopoiesis.

While dietary folate deficiency is associated with increased risk for birth defects and other diseases, evidence suggests that supplementation with folic acid can contribute to predisposition to some diseases, including immune dysfunction and cancer. Herein, we show that diets supplemented with folic acid both below and above the recommended levels led to significantly altered metabolism in multiple tissues in mice. Surprisingly, both low and excessive dietary folate induced similar metabolic changes, which were particularly evident for nucleotide biosynthetic pathways in B-progenitor cells.

Suppressor of Fused Plays an Important Role in Regulating Mesodermal Differentiation of Murine Embryonic Stem Cells In Vivo.

The hedgehog (Hh) signaling pathway plays fundamental roles during embryonic development and tumorigenesis. Previously, we have shown that ablation of the tumor suppressor and negative regulator, Suppressor of fused (Sufu), within this pathway causes embryonic lethality around E9.5 in the mouse.

Induced pluripotent stem cells in dermatology: potentials, advances, and limitations.

The discovery of methods for reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs) has raised the possibility of producing truly personalized treatment options for numerous diseases. Similar to embryonic stem cells (ESCs), iPSCs can give rise to any cell type in the body and are amenable to genetic correction by homologous recombination. These ESC properties of iPSCs allow for the development of permanent corrective therapies for many currently incurable disorders, including inherited skin diseases, without using embryonic tissues or oocytes.

Identification of a common Wnt-associated genetic signature across multiple cell types in pulmonary arterial hypertension.

Understanding differences in gene expression that increase risk for pulmonary arterial hypertension (PAH) is essential to understanding the molecular basis for disease. Previous studies on patient samples were limited by end-stage disease effects or by use of nonadherent cells, which are not ideal to model vascular cells in vivo. These studies addressed the hypothesis that pathological processes associated with PAH may be identified via a genetic signature common across multiple cell types.